U.S. patent application number 13/326792 was filed with the patent office on 2012-08-02 for regulatory t cell mediator proteins and uses thereof.
Invention is credited to David Gondek, Li-Fan Lu, Randolph J. Noelle, Sergio Quezada.
Application Number | 20120195894 13/326792 |
Document ID | / |
Family ID | 37215328 |
Filed Date | 2012-08-02 |
United States Patent
Application |
20120195894 |
Kind Code |
A1 |
Noelle; Randolph J. ; et
al. |
August 2, 2012 |
REGULATORY T CELL MEDIATOR PROTEINS AND USES THEREOF
Abstract
The present invention relates to novel regulatory T cell
proteins. One protein, designated PD-L3, resembles members of the
PD-L1 family, and co-stimulates .alpha.CD3 proliferation of T cells
in vitro. A second, TNF-like, protein has also been identified as
being upregulated upon .alpha.CD3/.alpha.GITR stimulation. This
protein has been designated T.sup.reg-sTNF. Proteins, antibodies,
activated T cells and methods for using the same are disclosed
Inventors: |
Noelle; Randolph J.;
(Plainfield, NH) ; Lu; Li-Fan; (Seattle, WA)
; Quezada; Sergio; (New York, NY) ; Gondek;
David; (Brookline, MA) |
Family ID: |
37215328 |
Appl. No.: |
13/326792 |
Filed: |
December 15, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11912397 |
Jun 4, 2008 |
|
|
|
PCT/US2006/015239 |
Apr 24, 2006 |
|
|
|
13326792 |
|
|
|
|
60674567 |
Apr 25, 2005 |
|
|
|
Current U.S.
Class: |
424/134.1 ;
424/133.1; 424/135.1; 424/173.1 |
Current CPC
Class: |
A01K 67/0278 20130101;
A61P 31/00 20180101; A61P 37/00 20180101; C07K 14/4702 20130101;
A61K 38/1709 20130101; G01N 33/505 20130101 |
Class at
Publication: |
424/134.1 ;
424/173.1; 424/133.1; 424/135.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 31/00 20060101 A61P031/00 |
Claims
1-6. (canceled)
7. A method of treating infection by potentiating T cell immunity
against the infectious gent comprising administering an effective
amount of an anti-PD-L3 antibody or antibody fragment that
specifically binds to the extracellular region of the human PD-L3
protein having the sequence in SEQ ID NO:4 or a soluble fusion
protein comprising a PD-L3 polypeptide that is a least 90%
identical to the extracellular domain of the PD-L3 protein having
the sequence in SEQ ID NO:3 or a fragment thereof which is at least
100 amino acids, wherein said anti-PD-L3 antibody or antibody
fragment antagonizes the immunosuppressive effect of PD-L3 on
immune cells in vivo, and thereby potentiates an immune response
against the infectious agent.
8. The method of claim 7, wherein the anti-PD-L3 antibody, antibody
fragment or fusion protein antagonizes one or more of the following
effects of PD-L3 in vivo: (1) suppression of T cell activation or
differentiation; (2) suppression of CD4+ T cell proliferation, (3)
suppression of CD8+ T cell proliferation and (4) suppression of
cytokine production by T cells.
9. The method of claim 7, wherein the antibody or antibody fragment
is a chimeric, human or humanized antibody or fragment thereof.
10. The method of claim 7, wherein the antibody fragment is
selected from a Fab, F(ab')2, Fv, Fd, and a scFv.
11. The method of claim 7, wherein the antibody is an IgG1.
12. The method of claim 7, which promotes NK cell or T
cell-mediated killing of infected cells.
Description
INTRODUCTION
[0001] This application claims benefit of U.S. Provisional
[0002] Patent Application Ser. No. 60/674,567, filed Apr. 25, 2005,
the content of which is incorporated herein by reference in its
entirety.
BACKGROUND OF THE INVENTION
[0003] Induction of an immune response requires T cell expansion,
differentiation, contraction and establishment of T cell memory. T
cells must encounter antigen presenting cells (APCs) and
communicate via T cell receptor (TCR)/major histocompatibility
complex (MHC) interactions on APCs. Once the TCR/MHC interaction is
established, other sets of receptor-ligand contacts between the T
cell and the APC are required, i.e. co-stimulation via CD154/CD40
and CD28/B7.1-B7.2. The synergy between these contacts is suggested
to result, in vivo, in a productive immune response capable of
clearing pathogens and tumors, and in some cases capable of
inducing autoimmunity.
[0004] Another level of control has been identified, namely
regulatory T cells (T.sup.reg). This specific subset of T cells is
generated in the thymus, delivered into the periphery, and is
capable of constant and inducible control of T cells responses in
vitro and in vivo (Sakaguchi (2000) Cell 101(5):455-8; Shevach
(2000) Annu. Rev. Immunol. 18:423-49; Bluestone and Abbas (2003)
Nat. Rev. Immunol. 3(3):253-7). T.sup.reg are represented by a
CD4.sup.+CD25.sup.+ phenotype and also express high levels of
cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), OX-40, 4-1BB
and the glucocorticoid inducible TNF receptor-associated protein
(GITR)(McHugh, et al. (2002) Immunity 16(2):311-23; Shimizu, et al.
(2002) Nat. Immun. 3(2):135-42). Elimination of T.sup.reg cells by
5 day neonatal thymectomy or antibody depletion using anti-CD25,
results in the induction of autoimmune pathology and exacerbation
of T cells responses to foreign and self-antigens, including
heightened anti-tumor responses (Sakaguchi, et al. (1985) J. Exp.
Med. 161(1):72-87; Sakaguchi, et al. (1995) J. Immunol.
155(3):1151-64; Jones, et al. (2002) Cancer Immun. 2:1). In
addition, T.sup.reg have also been involved in the induction and
maintenance of transplantation tolerance (Hara, et al. (2001) J.
Immunol. 166(6):3789-3796; Wood and Sakaguchi (2003) Nat. Rev.
Immunol. 3:199-210), since depletion of T.sup.reg with anti-CD25
monoclonal antibodies results in ablation of transplantation
tolerance and rapid graft rejection (Jarvinen, et al. (2003)
Transplantation 76:1375-9). Among the receptors expressed by
T.sup.reg, GITR seems to be an important component since in vitro
or in vivo ligation of GITR on the surface of T.sup.reg with an
agonistic monoclonal antibody results in rapid termination of
T.sup.reg activity (McHugh, at al. (2002) supra; Shimizu, et al.
(2002) supra), also resulting in autoimmune pathology (Shimizu, at
al. (2002) supra) and ablation of transplantation tolerance.
[0005] DNA microarray analysis has been conducted with a population
of T.sup.reg to identify genes differentially expressed by
T.sup.reg (Gavin, et al. (2002) Nat. Immunol. 3(1):33-41; McHugh,
at al. (2002) supra). The expression pattern of genes of
CD4.sup.+CD25.sup.- and CD4.sup.+CD25.sup.+ T cells was compared
(Gavin, et al. (2002) supra) as was the expression pattern of these
two populations of cells after activation by anti-CD3 antibody and
IL-2 for 12 and 48 hours (McHugh, et al. (2002) supra). However,
gene regulation by GITR signaling was not assessed.
[0006] T cell activation is dependent upon signs transferred
through antigen-specific T cells receptor recognition and accessory
receptors on the T cell. As the maintenance of immunologic
peripheral homeostatis is regulated by co-stimulatory molecules,
which play a critical role in suppressing autoreactive lymphocytes,
identification of these co-stimulatory molecules is needed.
[0007] A novel T cell co-stimulatory molecule has now been
identified and will be useful in modulating immune responses in
autoimmunity, cancer, infectious disease and transplantation.
SUMMARY OF THE INVENTION
[0008] The present invention is a composition containing an
isolated PD-L3 protein comprising the amino acid sequence set forth
in SEQ ID NO:5 and a pharmaceutically acceptable carrier. In one
embodiment, the PD-L3 protein is operably linked to a heterologous
protein.
[0009] The present invention is also an expression vector harboring
an isolated nucleic acid encoding PD-L3 protein comprising the
amino acid sequence set forth in SEQ ID NO:5; and host cells
containing said vector.
[0010] The present invention is also an isolated binding agent
which specifically binds to a PD-L3 protein comprising the amino
acid sequence set forth in SEQ ID NO:5.
[0011] The present invention is further a method for modulating an
immune cell response by contacting an immune cell with a PD-L3
protein, or binding agent thereof, in the presence of a primary
signal so that a response of the immune cell is modulated.
DETAILED DESCRIPTION OF THE INVENTION
[0012] A novel member of the PD-L1 family has now been identified
in T.sup.reg cells. This novel protein has been designated PD-L3.
Like other members of the PD-L1 family, PD-L3 co-stimulates
.alpha.CD3 proliferation of T cells in vitro. In addition, the
expression of PD-L3 is increased in .alpha.CD3 activated T.sup.reg
and reduced in the presence of .alpha.GITR. A second, TNF-like,
protein has also been identified as being upregulated upon
.alpha.CD3/.alpha.GITR stimulation. This protein has been
designated T.sup.reg-sTNF. These proteins may be involved in
contact-dependent and paracrine suppression of immunity and would
therefore be useful for modulating (e.g., inhibiting or
stimulating) an immune response and in the treatment of diseases
and conditions involving T.sup.reg signaling.
[0013] PD-L3 and T.sup.reg-sTNF were identified by global
transcriptional profiling of resting T.sup.reg, T.sup.reg activated
with .alpha.CD3, and T.sup.reg activated with
.alpha.CD3/.alpha.GITR. .alpha.GITR was selected for this analysis
as triggering of GITR on T.sup.reg has been shown to extinguish
their contact-dependent suppressive activity (Shimizu, et al.
(2002) supra). PD-L3 and T.sup.reg-sTNF were identified on
AFFIMETRIX.RTM. DNA arrays based on their unique expression
patterns (Table 1). PD-L3 exhibited an increase in expression in
.alpha.CD3 activated T.sup.reg and reduced expression in the
presence of .alpha.GITR; and T.sup.reg-sTNF exhibited a
.alpha.CD3/.alpha.GITR-dependent increase in expression.
TABLE-US-00001 TABLE 1 Relative Expression mRNA None .alpha.CD3
.alpha.CD3/.alpha.GITR PD-L3 6 10 7 T.sup.reg-sTNF 0.2 0.3 1.5
Purified CD4.sup.+CD25.sup.+ T cells were stimulated in culture
overnight with none, .alpha.CD3, or .alpha.CD3/.alpha.GITR, and RNA
isolated for real-time PCR analysis. Expression listed is relative
to actin.
[0014] PD-L3 was cloned and sequenced and, as indicated, is a
member of the PD-L1 family. This Ig family of co-stimulatory
molecules is composed of positive regulatory co-receptors such as
CD28 and ICOS, and also negative regulatory signals such as those
mediated by CTLA-4, PD-1, and BTLA molecules. Knockout studies of
negative co-stimulatory receptors have demonstrated the necessity
of these receptors in controlling autoimmunity and establishing
peripheral tolerance (Chen (2004) Nat. Rev. Immunol. 4:336-47). The
receptors of the PD-L1 family are type I transmembrane proteins
containing a single IgV domain while the ligands are type I
transmembrane proteins having both an IgV and an IgC extracellular
domain.
[0015] Sequence analysis revealed that PD-L3 corresponded to mouse
locus Ricken ID 4632428N05 with an mRNA coding sequence given as
GENBANK accession number NM.sub.--028732 and protein sequence give
as NP.sub.--080401. The nucleic acid sequence encoding mouse PD-L3
is set forth herein as SEQ ID NO:1 and the mouse PD-L3 protein
sequence is set forth as SEQ ID NO:2. PD-L3 has an Ig domain which
shares 26.5% homology with that of PD-L1 (B7-H1). The mouse PD-L3
gene is located on chromosome 10 (62.2 Mb) and is composed of 6
exons, creating a transcript of 4799 bases in length and coding for
a 309-residue type I transmembrane protein. Pfam and Interpro
(Integrated resource of Protein Families, Domains and Sites)
predict a signal sequence (positions 1-32) and an Ig-like domain
(positions 47-147). The human homolog of PD-L3 is located on
chromosome 10 (72.9 Mb) and composed of 6 exons thereby generating
a transcript of 4689 bases in length coding for a 311 residue
protein. The human homolog mRNA coding sequence is provided in
GENBANK accession number NM.sub.--022153 and protein sequence give
as NP.sub.--071436. The nucleic acid sequence encoding human PD-L3
is set forth herein as SEQ ID NO:3 and the human PD-L3 protein
sequence is set forth as SEQ ID NO:4. Mouse and human genes share
74% homology and are 68% identical at the protein level. Homologs
were also identified in Rattus norvegicus on chromosome 20 (27.7
Mb; GENBANK accession number BC098723), as well as Fugu rubripes
and Danio rerio. In particular embodiments, PD-L3 proteins of the
present share the common amino acid sequence set forth in SEQ ID
NO:5.
[0016] A PD-L3-Ig fusion protein was produced according to standard
methods, purified and titered into cultures of purified CD4.sup.+ T
cells, APC and .alpha.CD3. On day 3, all wells were pulsed with
tritiated thymidine (.sup.3H-TdR) and proliferation was determined.
Like other PD-L1 proteins, PD-L3 was shown to co-stimulate
.alpha.CD3 proliferation of T cells in vitro (Table 2).
TABLE-US-00002 TABLE 2 Treatment Proliferation (cpm/culture)
.alpha.CD3 (0.1 .mu.g/mL) 900 .+-. 600 hIgG1 (10 .mu.g/mL) 600 .+-.
400 PD-L3-Fc (0.5 .mu.g/mL) 2300 .+-. 1150 PD-L3-Fc (1.0 .mu.g/mL)
3900 .+-. 900 PD-L3-Fc (10 .mu.g/mL) 4200 .+-. 650
[0017] Using a rabbit anti-PD-L3 antibody, PD-L3 protein was
localized to lymphoid organs and prominently found in brain tissue.
Further, four transgenic mice were produced which expressed
full-length PD-L3 under the control of the human elongation factor
1 promoter. These mice were generated using lentiviral vector pWPT.
Similar to other PD-L1 family members (Appay, et al. (2002) J.
Immunol. 168:5954-8), it is contemplated that PD-L3 will function
as a negative regulator in vivo while functioning to co-stimulate
.alpha.CD3 T cell proliferation in vitro.
[0018] The second co-stimulatory molecule identified,
T.sup.reg-sTNF, contains a TNF-like domain similar to those found
in Clq family of proteins. Sequence analysis revealed that
T.sup.reg-sTNF corresponded with mouse locus Ricken ID 1110035L05
with an mRNA coding sequence given as GENBANK accession number
NM.sub.--026125 and protein sequence give as NP.sub.--080401. The
nucleic acid sequence encoding mouse T.sup.reg-sTNF is set forth
herein as SEQ ID NO:6 and the mouse T.sup.reg-sTNF protein sequence
is set forth herein as SEQ ID NO:7. This TNF-like molecule is
located on chromosome 4 (154.1 Mb), near OX40 and GITR, and
composed of 8 exons, creating a transcript 1301 bases in length
coding for a 308 residue soluble protein. Pfam and Interpro protein
predict a signal sequence (positions 1-19), a proline rich collagen
triple helix-like motif (positions 99-111), and a TNF-like motif
(positions 176-306). Collectively, these motifs are similar to
those of the Clq family of proteins, although this TNF-like protein
does not contain the characteristic Clq-like motif that identifies
this family. The human homolog of T.sup.reg-sTNF is located on
chromosome 1 (1.1 Mb) and is composed of 7 exons thereby generating
a transcript of 1014 bases in length coding for a 337 residue
protein. The human coding sequence for the human homolog of
T.sup.reg-sTNF is provided as GENBANK accession number BC089443 and
protein sequence give as AAH89443.1. The nucleic acid sequence
encoding human T.sup.reg-sTNF is set forth herein as SEQ ID NO:8
and the human T.sup.reg-sTNF protein sequence is set forth as SEQ
ID NO:9. Mouse and human genes share 65.3% homology and 66%
identify at the protein level. Homologs were also identified in
Rattus norvegicus on chromosome 5 (172.8 Mb; GENBANK accession
number XM.sub.--233720.2), as well as Fugu rubripes and Danio
rerio. In particular embodiments, T.sup.reg-sTNF proteins of the
present share the common amino acid sequence set forth in SEQ ID
NO:10.
[0019] Having identified a novel immune cell regulatory molecule
produced by T.sup.reg cells, the present invention relates to a
PD-L3 protein, agents which bind PD-L3, nucleic acids encoding
PD-L3 and methods of using PD-L3 and PD-L3 binding agents to
modulate immune cell responses.
[0020] As used herein, a PD-L3 protein is intended to include a
protein that has a sequence which is substantially similar to that
of mouse PD-L3 (i.e., SEQ ID NO:2) or human PD-L3 (i.e., SEQ ID
NO:4) and in particular embodiments has the consensus amino acid
sequence set forth in SEQ ID NO:5. The term substantially similar
refers to sequences having sequence variation (e.g., conservative
substitutions and/or variations) that do not materially affect the
nature of the protein (i.e., the structure, stability
characteristics, substrate specificity and/or biological activity
of the protein). In general, a protein having an amino acid
sequence that is substantially similar to SEQ ID NO:2 or SEQ ID
NO:4 has at least 70% identity to that of SEQ ID NO:2 or SEQ ID
NO:4, over its entire length and exhibits at least one biological
activity of PD-L3. The present invention further provides for a
protein which has an amino acid sequence which shares at least 80%
identity, at least 90% identity, at least 95% identity, or more
desirably at least 97-99% identity, to that of SEQ ID NO:2 or SEQ
ID NO:4 over the entire length of SEQ ID NO:2 or SEQ ID NO:4.
[0021] Percent identical and percent similar are used herein in
comparisons among amino acid and nucleic acid sequences. When
referring to amino acid sequences, identity or percent identical
refers to the percent of the amino acids of the subject amino acid
sequence that have been matched to identical amino acids in the
compared amino acid sequence by a sequence analysis program.
Percent similar refers to the percent of the amino acids of the
subject amino acid sequence that have been matched to identical or
conserved amino acids. Conserved amino acids are those which differ
in structure but are similar in physical properties such that the
exchange of one for another would not appreciably change the
tertiary structure of the resulting protein. Conservative
substitutions are well-known in the art (see, e.g., Taylor (1986)
J. Theor. Biol. H 9:205). When referring to nucleic acid molecules,
percent identical refers to the percent of the nucleotides of the
subject nucleic acid sequence that have been matched to identical
nucleotides by a sequence analysis program.
[0022] Identity and similarity can be readily calculated by known
methods. Nucleic acid sequences and amino acid sequences can be
compared using computer programs that align the similar sequences
of the nucleic or amino acids thus define the differences. Such
methods include the BLAST programs (NCBI) and the DNAstar system
(Madison, Wis.). However, equivalent alignments and
similarity/identity assessments can be obtained through the use of
any standard alignment software. For instance, the GCG Wisconsin
Package, available from the Genetics Computer Group in Madison,
Wis., can also be used to compare sequence identity and
similarity.
[0023] A PD-L3 protein can be in the form of a mature protein (i.e.
lacking a signal sequence, residues 1-32 of SEQ ID NO:1 or SEQ ID
NO:2) or can be a part of a larger protein such as a fusion protein
(e.g., fused to Fc). It is often advantageous to also include amino
acid sequences which contain secretory or leader sequences,
pro-sequences, sequences which aid in purification such as multiple
histidine residues, or an additional sequence for stability during
recombinant production. Accordingly, one embodiment of the present
invention is a mature PD-L3 protein lacking N-terminal signal
sequences. Another embodiment of the present invention is a fusion
protein composed of PD-L3, or a fragment thereof, operably linked
to a heterologous peptide or polypeptide (e.g., GST, Ig, His.sub.6,
and the like) such that the fused proteins are translated in-frame.
As used herein, a heterologous peptide or protein is one which is
not naturally found to be operably linked to PD-L3.
[0024] A particular suitable heterologous peptide is an
immunoglobulin constant region, for example, a human C.gamma.1
domain or C.gamma..sub.4 domain (e.g., the hinge, CH2 and CH3
regions of human IgC.gamma.1, or human IgC.gamma.4; see e.g.,
Capon, et al. U.S. Pat. Nos. 5,116,964; 5,580,756; 5,844,095 and
the like). Such constant regions may retain regions which mediate
effector function (e.g., Fc receptor binding) or may be altered to
reduce effector function.
[0025] Fragments of a PD-L3 protein are also included in the
invention. A fragment is a protein having an amino acid sequence
that is entirely the same as part, but not all, of the amino acid
sequence of the aforementioned PD-L3 protein. Fragments include,
for example, truncation polypeptides having the amino acid sequence
of a PD-L3 protein, except for deletion of a continuous series of
residues that includes the amino terminus, or a continuous series
of residues that includes the carboxyl terminus or deletion of two
continuous series of residues, one including the amino terminus and
one including the carboxyl terminus. Other fragments are
biologically active fragments. Biologically active fragments are
those that mediate PD-L3 activity (e.g., co-stimulation of T cells
or modulation of an immune response), including those with a
similar activity or an improved activity, or with a decreased
undesirable activity. Also included are those that are antigenic or
immunogenic in an animal.
[0026] A PD-L3 protein of the invention can be prepared in any
suitable manner. If produced in situ, the protein can be purified
from appropriate sources, e.g., appropriate vertebrate cells e.g.,
mammalian cells for instance T.sup.reg cells from human, mouse,
bovine or rat.
[0027] Alternatively, the availability of nucleic acid molecules
encoding the PD-L3 protein enables production of PD-L3 using in
vitro expression methods known in the art. For example, a cDNA or
gene can be cloned into an appropriate in vitro transcription
vector, for in vitro transcription, followed by cell-free
translation in a suitable cell-free translation system. In vitro
transcription and translation systems are commercially available,
e.g., from PROMEGA.RTM. Biotech, Madison, Wis., or GIBCO-BRL.RTM.,
Rockville, Md. In vitro transcription and translation is suitable
for preparing small amounts of native or mutant proteins for
research purposes, particularly since it allows the incorporation
of radioactive nucleotides.
[0028] Larger quantities of PD-L3 protein can be produced by
expression in a suitable prokaryotic or eukaryotic system. For
example, part or all of a DNA molecule, such as the coding portion
of SEQ ID NO:1 or SEQ ID NO:3 can be inserted into a plasmid vector
adapted for expression in a bacterial cell (such as Escherichia
coli) or a yeast cell (such as Saccharomyces cerevisiae), or into a
baculovirus vector for expression in an insect cell. Such vectors
contain the regulatory elements necessary for expression of the DNA
in the host cell, positioned in such a manner as to permit
expression of the DNA into mRNA and mRNA into protein in the host
cell. Such regulatory elements required for expression include
promoter sequences, transcription initiation sequences and,
optionally, enhancer sequences. Suitable vectors for recombinant
protein expression in mammalian, yeast, or prokaryotic systems are
commercially available from such sources as STRATAGENE.RTM.,
INVITROGEN.TM., Pharmacia and the like.
[0029] Host-specific secretion signals can be used to facilitate
purification of the resulting protein. The coding sequence for the
secretion peptide is operably linked to the 5' end of the coding
sequence for the protein, and this hybrid nucleic acid molecule is
inserted into a plasmid adapted to express the protein in the host
cell of choice. Plasmids specifically designed to express and
secrete foreign proteins are available from commercial sources. For
example, if expression and secretion is desired in E. Coli,
commonly used plasmids include pTrcPPA (Pharmacia); pPROK-C and
pKK233-2 (CLONTECH.TM.); and pNH8a, pNH16a, pcDNAII and pAX
(STRATAGENE.RTM.), among others.
[0030] A PD-L3 protein produced by in vitro transcription and
translation or by gene expression in a recombinant prokaryotic or
eukaryotic system can be purified according to methods known in the
art (e.g., fractionation on immunoaffinity or ion-exchange columns;
ethanol precipitation; reverse phase HPLC; chromatography on silica
or on a cation-exchange resin such as DEAE; chromatofocusing;
SDS-PAGE; ammonium sulfate precipitation; or gel filtration using,
for example, SEPHADEX.RTM. G-75).
[0031] Alternatively, a synthetic PD-L3 protein can be prepared
using various synthetic methods of peptide synthesis via
condensation of one or more amino acid residues, in accordance with
conventional peptide synthesis methods. For example, peptides are
synthesized according to standard solid-phase methodologies, such
as may be performed on an APPLIED BIOSYSTEMS.TM. Model 430A peptide
synthesizer (APPLIED BIOSYSTEMS.TM., Foster City, Calif.),
according to manufacturer's instructions. Other methods of
synthesizing peptides or peptidomimetics, either by solid phase
methodologies or in liquid phase, are well-known to those skilled
in the art.
[0032] PD-L3 peptidomimetics (Fauchere, J. (1986) Adv. Drug Res.
15:29; Veber and Freidinger (1985) TINS p. 392; and Evans et al.
(1987) J. Med. Chem. 30:1229) are also contemplated.
Peptidomimetics are usually developed with the aid of computerized
molecular modeling. Peptide mimetics that are structurally similar
to therapeutically useful peptides can be used to produce an
equivalent therapeutic or prophylactic effect. Generally,
peptidomimetics are structurally similar to a paradigm polypeptide
(i.e., a polypeptide that has a biological or pharmacological
activity), such as human PD-L3, but have one or more peptide
linkages optionally replaced by a linkage such as --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and
trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by
methods known in the art (see, e.g., Spatola (1983) In: Chemistry
and Biochemistry of Amino Acids, Peptides, and Proteins, Weinstein,
B., ed., Marcel Dekker, New York, p. 267; Morley (1980) Trends
Pharm. Sci. pp. 463-468; Hudson, et al. (1979) Int. J. Pept. Prot.
Res. 14:177-185); Spatola, et al. (1986) Life Sci. 38:1243-1249;
Hann (1982) J. Chem. Soc. Perkin Trans. I. 307-314; Jennings-White,
et al. (1982) Tetrahedron Lett. 23:2533; Holladay, et al. (1983)
Tetrahedron Lett. (1983) 24:4401-4404; Hruby (1982) Life Sci.
(1982) 31:189-199).
[0033] Whether recombinantly-produced or chemically-synthesized,
PD-L3 (including PD-L3 fusion proteins or biologically active PD-L3
fragments) can be formulated into a pharmaceutically acceptable
composition for use in accordance with the methods disclosed
herein. A PD-L3 protein is generally formulated with a
pharmaceutically acceptable carrier, such as buffered saline; a
polyol (e.g., glycerol, propylene glycol, liquid polyethylene
glycol and the like); carbohydrates such as glucose, mannose,
sucrose or dextrans, mannitol; amino acids such as glycine;
antioxidants; chelating agents such as EDTA or glutathione;
preservatives or suitable mixtures thereof. In addition, a
pharmaceutically acceptable carrier can include any solvent,
dispersion medium, and the like which may be appropriate for a
desired route of administration of the composition. The use of such
carriers for pharmaceutically active substances is known in the
art. Suitable carriers and their formulation are described, for
example, in Remington: The Science and Practice of Pharmacy,
Alfonso R. Gennaro, editor, 20th ed. Lippingcott Williams &
Wilkins: Philadelphia, Pa., 2000.
[0034] A PD-L3 protein of the present invention can be used to
identify binding partners of PD-L3, i.e., binding agents and
receptors. In these assays, PD-L3 is allowed to form a physical
interaction with the unknown binding partner(s), often in a
heterologous solution of molecules. The binding complex is then
isolated, and the identity of the binding partner is determined
(e.g., via mass spec or sequence analysis). Alternatively, a panel
of rational binding partners (e.g., CTLA-4, PD-1, or BTLA) can be
screened with a PD-L3 protein. These procedures are greatly
facilitated by simple methods for isolating PD-L3 protein, e.g.,
precipitation using immunologically-specific antibodies to the
PD-L3 protein, or purification with PD-L3 protein bound to a solid
support. In one embodiment, a PD-L3 protein is attached to a solid
support via a covalent linkage. In other embodiments, attachment is
via a non-covalent linkage, for example, between members of a high
affinity binding pair (e.g., ligand/receptor or antigen/antibody
pairs). Suitable solid supports include beads, e.g., magnetized
beads or beads which are dense enough to be separated form
non-associated protein by centrifugation. Alternatively, the PD-L3
protein can be used in a yeast two hybrid system such as the
Ga14/LacZ system (see Clark, et al. (1998) Proc. Natl. Acad. Sci.
USA 95:5401-5406) to identify binding partners.
[0035] A PD-L3 protein of the present invention can also be used as
a regulatory signal in a method for modulating, i.e., stimulating
or inhibiting, an immune cell response. The prototypic immune
response described herein is stimulation of T cells (CD4.sup.+),
but one of ordinary skill in the art will readily appreciate that
the method can be applied to modulation of other T cell-mediated
and/or B cell-mediated immune responses that are influenced by
modulation of T cell co-stimulation. By way of example, immune
responses of tumor-reactive lymphocytes (CD8.sup.+; Hellstrom, et
al. (2001) Proc. Natl. Acad. Sci. USA 98:6783-6788), CD43.sup.+ T
cells (Wang, et al. (2004) J. Immunol. 173(10):6294-302), and
natural killer cells can be modulated. In addition, immune
responses that are indirectly effected by T cell activation, e.g.,
antibody production (humoral responses) and activation of cytokine
responsive cells, e.g., macrophages are also contemplated.
[0036] In one embodiment, a PD-L3 protein is used as a
co-stimulatory signal for stimulating or enhancing immune cell
activation. A co-stimulatory signal, as used herein, refers to a
signal, which in combination with a primary signal, such as TCR/CD3
ligation, leads to immune cell activation. The co-stimulatory
signal can be used simultaneously with or subsequent to the primary
signal to achieve the desired result. The term activation, within
the context of T cells, refers to the induction of cellular
proliferation. Activation of a T cell may also induce cytokine
production and performance of regulatory or cytolytic effector
functions. T cell activation preferably results in at least a 2.5-
to 4.5-fold increase in the cell population as compared to cells
which have not been co-stimulated (i.e., no stimulation or
stimulated with a primary signal only). T cell activation can be
quantitated as exemplified herein via tritiated thymidine
incorporation or by analyzing cytokine production. Cytokines can be
measured according to biological activity or protein accumulation
(e.g., as determined by various immuno-based, activity, or other
assays). Alternatively, mRNA production can be measured to
establish levels of stimulation of transcription. In particular
embodiments, the primary and co-stimulatory signals are further
used in combination with other agents, such as cytokines (IL-2,
IL-4, IL-7, IL-10, IL-12, etc.) or antigen presenting cells, for
optimal activation.
[0037] In another embodiment, a PD-L3 protein is used as an
inhibitory signal for inhibiting or decreasing immune cell
activation. In this embodiment, the inhibitory signal binds to an
inhibitory receptor (e.g., CTLA4 or PD-1) on an immune cell thereby
antagonizing the primary signal which binds to an activating
receptor (e.g., via a TCR, CD3, BCR, or Fc polypeptide). Inhibition
includes, e.g., inhibition of second messenger generation; an
inhibition of proliferation; an inhibition of effector function in
the immune cell, e.g., reduced phagocytosis, reduced antibody
production, reduced cellular cytotoxicity, the failure of the
immune cell to produce mediators, (such as cytokines (e.g., IL-2)
and/or mediators of allergic responses); or the development of
anergy.
[0038] In particular embodiments, the primary signal is a ligand
(e.g., CD3 or anti-CD3) that binds TCR and initiates a primary
stimulation signal. Such TCR ligands are readily available from
commercial sources and specific examples include anti-CD3 antibody
OKT3, prepared from hybridoma cells obtained from the American Type
Culture Collection, and anti-CD3 monoclonal antibody G19-4. In an
alternative embodiment, a primary signal is delivered to a T cell
through other mechanisms including a protein kinase C activator,
such as a phorbol ester (e.g., phorbol myristate acetate), and a
calcium ionophore (e.g., ionomycin, which raises cytoplasmic
calcium concentrations), or the like. The use of such agents
bypasses the TCR/CD3 complex but delivers a stimulatory signal to T
cells. Other agents acting as primary signals can include natural
and synthetic ligands. A natural ligand can include MHC with or
without a peptide presented. Other ligands can include, but are not
limited to, a peptide, polypeptide, growth factor, cytokine,
chemokine, glycopeptide, soluble receptor, steroid, hormone,
mitogen, such as PHA, or other superantigens, peptide-MHC tetramers
(Altman, et al. (1996) Science 274(5284):94-6) and soluble MHC
dimers (Dal Porto, et al (1993) Proc. Natl. Acad. Sci. USA 90:
6671-5).
[0039] Immune cells activated in accordance with the method of the
instant invention can subsequently be expanded ex vivo and used in
the treatment and prevention of a variety of diseases; e.g., human
T cells which have been cloned and expanded in vitro maintain their
regulatory activity (Groux, et al. (1997) Nature 389(6652):737-42).
Prior to expansion, a source of T cells is obtained from a subject
(e.g., a mammals such as a human, dog, cat, mouse, rat, or
transgenic species thereof). T cells can be obtained from a number
of sources, including peripheral blood mononuclear cells, bone
marrow, lymph node tissue, cord blood, thymus tissue, tissue from a
site of infection, spleen tissue, tumors or T cell lines. T cells
can be obtained from a unit of blood collected from a subject using
any number of techniques known to the skilled artisan, such as
FICOLL.TM. separation.
[0040] Alternatively, T cells from the circulating blood of an
individual are obtained by apheresis or leukapheresis. The
apheresis product typically contains lymphocytes, including T
cells, monocytes, granulocytes, B cells, other nucleated white
blood cells, red blood cells, and platelets. The cells collected by
apheresis are washed to remove the plasma fraction and to place the
cells in an appropriate buffer (e.g., phosphate buffered saline
(PBS) or wash solution lacking calcium or other divalent cations)
or media for subsequent processing steps. After washing, the cells
are resuspended in a variety of biocompatible buffers, such as,
calcium-free, magnesium-free PBS.
[0041] Isolation of T cells from peripheral blood lymphocytes can
be carried out by lysing the red blood cells and depleting the
monocytes, for example, by centrifugation through a PERCOLL.TM.
gradient. A specific subpopulation of T cells, such as CD28.sup.+,
CD4.sup.+, CD8.sup.+, CD45RA.sup.+, and CD45RO.sup.+ T cells, can
be further isolated by positive or negative selection techniques
well-known to the skilled. Enrichment of a T cell population by
negative selection can be accomplished with a combination of
antibodies directed to surface markers unique to the negatively
selected cells. For example, to enrich for CD4.sup.+ cells by
negative selection, a monoclonal antibody cocktail typically
includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and
CD8.
[0042] T cells for stimulation can also be frozen after isolation.
Many freezing solutions and parameters are known in the art and
will be useful in this context. One method involves suspending the
cells in PBS containing 20% DMSO and 8% human serum albumin, or
other suitable cell freezing media, and subsequently freezing the
cells at -80.degree. C. at a rate of 1.degree. C. per minute and
stored in the vapor phase of a liquid nitrogen storage tank. Other
methods of controlled freezing can be used as well as uncontrolled
freezing immediately at -20.degree. C. or in liquid nitrogen.
[0043] Those of ordinary skill in the art will readily appreciate
that stimulation and expansion of T cells described herein can be
carried out in a variety of environments (i.e., containers). For
example, such containers can be culture flasks, culture bags, or
any container capable of holding cells (e.g., a bioreactor),
preferably in a sterile environment. For example, several
manufacturers currently make devices that can be used to grow cells
and be used in combination with the methods of the present
invention. See for example, Celdyne Corp. (Houston, Tex.), Unisyn
Technologies (Hopkinton, Mass.), Synthecon, Inc. (Houston, Tex.),
Aastrom Biosciences, Inc. (Ann Arbor, Mich.), Wave Biotech LLC
(Bedminster, N.J.). Further, patents covering bioreactors include
U.S. Pat. Nos. 6,096,532; 5,985,653; 5,888,807; and 5,190,878.
[0044] The present invention also relates to a vector, in
particular an expression vector, containing nucleic acids encoding
a PD-L3 protein for therapeutic use and production of recombinant
PD-L3 protein. A PD-L3 nucleic acid of the present invention
includes nucleic acids encoding the PD-L3 protein containing the
amino acid sequence set forth in SEQ ID NO:5 and fragments, and
nucleic acids substantially similar thereto. More specifically,
PD-L3 nucleic acids of the invention include nucleic acids encoding
mouse and human PD-L3 protein, wherein such nucleic acids are set
forth in SEQ ID NO:1 and SEQ ID NO:3, respectively. A nucleic acid
that is substantially similar to a PD-L3 nucleic acid shares at
least 70% identity over its entire length with a nucleotide
sequence encoding a PD-L3 protein of SEQ ID NO:2 or SEQ ID NO:4,
and a nucleic acid having a nucleotide sequence that is at least
70% identical to that of SEQ ID NO: 1 or SEQ ID NO:3, over its
entire length. In particular embodiments, a nucleic acid that is
substantially similar to a PD-L3 nucleic acid shares at least 80%
identity, at least 90% identity, at least 95% identity, or more
desirably at least 97-99% identity, to that of SEQ ID NO:1 or SEQ
ID NO:3 over the entire length of SEQ ID NO:1 or SEQ ID NO:3. Also
encompassed within the scope of a PD-L3 nucleic acid is a
nucleotide sequence which has sufficient identity to a nucleotide
sequence contained in SEQ ID NO:1 or SEQ ID NO:3, to hybridize
under conditions useable for amplification, as a probe or marker,
or antisense or siRNA.
[0045] In accordance with the present invention, nucleic acids
having the appropriate level sequence homology (i.e., 70% identity
or greater) with part or all the coding regions of SEQ ID NO:1 or
SEQ ID NO:3 can be identified by using hybridization and washing
conditions of appropriate stringency. For example, hybridizations
can be performed, according to the method of Sambrook, et al.
((1989) Molecular Cloning, a Laboratory Manual, Cold Spring Harbor
Laboratories, New York) using a hybridization solution containing
1.0% SDS, up to 50% formamide, 5.times.SSC (150 mM NaCl, 15 mM
trisodium citrate), 0.05% sodium pyrophosphate (pH 7.6),
5.times.Denhardt's solution, and 100 microgram/mL denatured,
sheared salmon sperm DNA. Hybridization is carried out at
37-42.degree. C. for at least six hours. Following hybridization,
filters are washed as follows: 5 minutes at room temperature in
2.times.SSC and 1% SDS; 15 minutes at room temperature in
2.times.SSC and 0.1% SDS; 30 minutes to 1 hour at 37.degree. C. in
2.times.SSC and 0.1% SDS; and 2 hours at 45-55.degree. C. in
2.times.SSC and 0.1% SDS, changing the solution every 30
minutes.
[0046] One common formula for calculating the stringency conditions
required to achieve hybridization between nucleic acid molecules of
a specified percent identity is set forth by: T.sub.m=81.5.degree.
C.+16.6 log 10 ([Na+]/(1.0+0.7[Na+]))+0.7% GC-500/size (Wetmur
(1991) Crit. Rev. Biochem. Mol. Biol. 26:227-259)
[0047] The stringency of the hybridization and wash depend
primarily on the salt concentration and temperature of the
solutions. In general, to maximize the rate of annealing of the
probe with its target, the hybridization is usually carried out at
salt and temperature conditions that are 20-25.degree. C. below the
calculated T.sub.m of the of the hybrid. Wash conditions should be
as stringent as possible for the degree of identity of the probe
for the target. In regards to the nucleic acids of the present
invention, a moderate stringency hybridization is defined as
hybridization in 6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS
and 100 .mu.g/mL denatured salmon sperm DNA at 42.degree. C., and
wash in 2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes. A
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/mL
denatured salmon sperm DNA at 42.degree. C., and wash in
1.times.SSC and 0.5% SDS at 6-5.degree. C. for 15 minutes. Very
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times.Denhardt's solution, 0.5% SDS and 100 .mu.g/mL
denatured salmon sperm DNA at 42.degree. C., and wash in
0.1.times.. SSC and 0.5% SDS at 65.degree. C. for 15 minutes.
[0048] Oligonucleotides (sense or antisense strands of DNA, cDNA or
RNA) having sequences capable of hybridizing with at least one
sequence of a nucleic acid molecule encoding the PD-L3 protein are
useful as probes for detecting PD-L3 genes or transcripts and can
also be useful in the treatment of various diseases or conditions,
when delivered by an appropriate vehicle to the affected cells.
Oligonucleotides for use as probes, primers, antisense, or siRNA
are based on rationally-selected nucleic acid sequences chosen from
SEQ ID NO:1 or SEQ ID NO:3. Such oligonucleotides can be used for
the detection and isolation of nucleic acids encoding PD-L3 or
inhibition of PD-L3 expression. Further, amino acid sequences of
SEQ ID NO:2 and SEQ ID NO:4 can be used to design degenerate
oligonucleotide primers, as is commonly done by those skilled in
the art, for screening cDNA libraries from, e.g., bovine, canine,
and feline to obtain PD-L3 homologs from bovine, canine, and
feline, respectively.
[0049] The nucleotide sequences encoding the PD-L3 protein of SEQ
ID NO:2 or SEQ ID NO:4 can be identical to the protein encoding
sequence contained in SEQ ID NO:1 or SEQ ID NO:3, or can be a
sequence, which as a result of the redundancy (degeneracy) of the
genetic code, also encodes the protein of SEQ ID NO:2 or SEQ ID
NO:4.
[0050] When the nucleic acids of the invention are used for the
recombinant production of a PD-L3 protein, the nucleic acid can
include the coding sequence for the mature protein or a fragment
thereof, by itself; the coding sequence for the mature protein or
fragment in reading frame with other coding sequences, such as
those encoding a leader or secretory sequence, or other fusion
peptide or protein as discussed supra. For example, a marker
sequence which facilitates purification of the fused polypeptide
can be encoded. The PD-L3 nucleic acid can also contain non-coding
5' and 3' sequences, such as transcribed, non-translated sequences,
splicing and polyadenylation signals, ribosome binding sites and
sequences that stabilize mRNA.
[0051] Nucleic acids of the present invention can be maintained as
DNA in any convenient cloning vector, e.g., in plasmid
cloning/expression vector, such as pBLUESCRIPT.RTM.
(STRATAGENE.RTM.), that is propagated in a suitable E. coli host
cell. As described above, PD-L3 nucleic acids may be used to
produce large quantities of substantially pure PD-L3 proteins, or
selected fragments thereof.
[0052] Hence, the present invention also relates to vectors, in
particular expression vectors, that contain a PD-L3 nucleic acid,
and isolated host cells that are genetically engineered with said
vectors. Expression vectors harboring PD-L3 nucleic acids are
discussed supra and generally contain all the necessary regulatory
sequences, for example, promoter and terminator sequences, operably
linked to the PD-L3 nucleic acids such that the PD-L3 coding
sequence is transcribed into RNA and subsequently translated into
protein or in the case of antisense, transcribed into RNA. Large
numbers of suitable vectors and regulatory sequences are known to
those of skill in the art, and are commercially available. The
following vectors are provided by way of example, bacterial vectors
pQE70, pQE60, pQE-9 (QIAGEN.RTM.), pBS, pD10, pBLUESCRIPT.degree.
SK, pBSKS, pNH8A, pNHI8A, pNH.sub.46A (STRATAGENE.RTM.) and pRIT5
(Pharmacia); and eukaryotic vectors pWLNEO, pSV2CAT, pOG44, pXTI,
pSG (STRATAGENE.RTM.) pSVK3, pBPV, pMSG, pSVL (Pharmacia). As
further examples, a PD-L3 cDNA of can be inserted in the
pEF/myc/cyto vector (INVITROGEN.TM.) or the pCMV-Tag3b vector
(STRATAGENE.RTM.) and transformed (e.g., calcium phosphate
transfection, DEAE-dextran mediated transfection, microinjection,
cationic lipid-mediated transfection, electroporation) into Hela
thereby facilitating purification and use of PD-L3.
[0053] However, any other plasmid or vector can be used as long as
they are replicable and viable in the host. In addition, a complete
mammalian transcription unit and a selectable marker can be
inserted into a prokaryotic plasmid for use in in vivo procedures.
The resulting vector is then amplified in bacteria before being
transfected into cultured mammalian cells or delivered directly to
the subject with an acceptable carrier. Examples of vectors of this
type include pTK2, pHyg and pRSVneo. Hence, these plasmids,
constructs and vectors can be used in both in vivo and ex vivo
procedures. Ex vivo procedures involve the removal of a host cell
(e.g., a T.sup.reg cell) from a subject, recombinant manipulation
of the cell (i.e., transformation, transduction or transfection
with a suitable PD-L3 expression vector), and the re-delivery of
the cell back into its host environment.
[0054] Representative examples of appropriate hosts for in vitro
procedures include bacterial cells, such as streptococci,
staphylococci, E. coli, Streptomyces and Bacillus subtilis cells;
fungal cells, such as yeast cells and Aspergillus cells, insect
cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells
such as CHO, COS, HeLa, 0127, 3T3, BHK, and HEK 293 cells, and
plant cells. The selection of an appropriate host is deemed to be
within the scope of those skilled in the art from the teachings
herein.
[0055] Genetic material, such as the nucleic acids of the present
invention, can be delivered to cells, in vivo, using various
different plasmid-based delivery platforms, including but not
limited to recombinant ADV (such as that described in U.S. Pat. No.
6,069,134), AAV (such as those described by U.S. Pat. No.
5,139,941), MMLV, Herpes Simplex Virus (U.S. Pat. No. 5,288,641),
cytomegalovirus, lentiviral, and overall, retroviral gene delivery
systems, well-known and practiced with in the art.
[0056] Techniques for preparing replication defective, infective
viruses are well-known in the art (see, e.g., Gluzman et al. (1982)
Virology 123(1):78-92). These systems typically include a plasmid
vector including a promoter sequence (e.g., CMV immediate early,
HSV thymidine kinase, early and late SV40, LTRs from retrovirus,
and mouse metallothionein-1) operably linked to the nucleotide
coding the gene of interest (inserted into an appropriate gene
insertion site, i.e., an IRES site), as well as a terminating
signal (such as a poly-A tail, i.e., BGH), and the appropriate
mutations so as to make the delivery vehicle replication defective
(e.g., Psi sequence deletions) and safe for therapeutic uses. The
construction of the appropriate elements in a vector system
containing the nucleotides of the present invention is well within
the skills of one versed in the recombinant arts.
[0057] Therapeutic nucleic acids can be delivered to target cells
via basic transfection methods such as permeabilizing the cell
membrane physically or chemically. Liposomes or protein conjugates
formed with certain lipids and amphophilic peptides can also be
used for transfection (Stewart, et al. (1992) Hum. Gene Ther.
3(3):267-75; Zhu, et al. (1993) Science 261(5118):209-11). This
approach is particularly effective in ex vivo procedures involving
leukocytes, which can be temporarily removed from the body and can
tolerate the cytotoxicity of the treatment.
[0058] A second, transduction approach, capitalizes on the natural
ability of viruses to enter cells, bringing their own genetic
material with them. For example, retroviruses can integrate their
genes into the host genome, transferring a large amount of foreign
genetic material, infecting a broad spectrum of species and cell
types and of being packaged in special cell-lines (Miller (1992)
Curr. Top. Microbiol. Immunol. 158:1-24).
[0059] A third method uses other viruses, such as adenovirus,
herpes simplex viruses (HSV), cytomegalovirus (CMV), and
adeno-associated virus (AAV), which are engineered to serve as
vectors for gene transfer. For example, in an adenovirus gene
transfer systems recombinant engineered adenovirus is rendered
replication-incompetent by deletion of a portion of its genome,
such as E1, and yet still retains its competency for infection.
Relatively large foreign proteins can be expressed when additional
deletions are made in the adenovirus genome. For example,
adenoviruses deleted in both E1 and E3 regions are capable of
carrying up to 10 Kb of foreign DNA and can be grown to high titers
in 293 cells with persistent expression of transgenes following
adenoviral infection in vivo.
[0060] In addition to therapeutic uses and recombinant protein
production, vectors and host cells disclosed herein are useful for
producing transgenic animals which constitutively overexpress PD-L3
or are deficient in PD-L3 protein production (i.e., knock out
animals).
[0061] The present invention further relates to an isolated binding
agent which specifically recognizes and binds to a PD-L3 protein.
Binding agents are intended to include antibodies as well as
peptide aptamers.
[0062] Peptide aptamers which specifically bind to a PD-L3 protein
can be rationally designed or screened for in a library of aptamers
(e.g., provided by Aptanomics SA, Lyon, France). In general,
peptide aptamers are synthetic recognition molecules whose design
is based on the structure of antibodies. Peptide aptamers consist
of a variable peptide loop attached at both ends to a protein
scaffold. This double structural constraint greatly increases the
binding affinity of the peptide aptamer to levels comparable to
that of an antibody (nanomolar range).
[0063] An antibody to a PD-L3 protein can be generated using
methods that are well-known in the art. An anti-PD-L3 antibody is
intended to include a polyclonal and monoclonal antibody; humanized
antibody; murine antibody; mouse-human antibody; mouse-primate
antibody; and chimeric antibody; wherein the antibody can be an
intact molecule, a fragment thereof (such as scFv, Fv, Fd, Fab,
Fab' or F(ab)'.sub.2 fragment), or a multimer or aggregate of
intact molecules and/or fragments; and can occur in nature or be
produced, e.g., by immunization, synthesis or genetic engineering.
An antibody fragment, as used herein, refers to fragments, derived
from or related to an antibody, which bind antigen and which can be
derivatized to exhibit structural features that facilitate
clearance and uptake, e.g., by the incorporation of galactose
residues. This includes, e.g., F(ab), F(ab)'.sub.2, scFv, light
chain variable region (V.sub.L,), heavy chain variable region
(V.sub.H), and combinations thereof.
[0064] Monoclonal antibodies to PD-L3 protein of the invention can
be prepared using any technique which provides for the production
of antibody molecules by continuous cell lines in culture. These
include, but are not limited to, the hybridoma technique, the human
B-cell hybridoma technique, and the EBV-hybridoma technique
(Kohler, et al. (1975) Nature 256:495-497; Kozbor, et al. (1985) J.
Immunol. Methods 81:31-42; Cote, et al. (1983) Proc. Natl. Acad.
Sci. 80:2026-2030; Cole, et al. (1984) Mol. Cell. Biol.
62:109-120).
[0065] In addition, techniques developed for the production of
humanized and chimeric antibodies, the splicing of mouse antibody
genes to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used (Morrison,
et al. (1984) Proc. Natl. Acad. Sci. 81, 6851-6855; Neuberger, et
al. (1984) Nature 312:604-608; Takeda, et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single-chain antibodies can be adapted, using methods
known in the art, to produce specific, single-chain antibodies.
Antibodies with related specificity, but of distinct idiotypic
composition, can be generated by chain shuffling from random
combinatorial immunoglobulin libraries (Burton (1991) Proc. Natl.
Acad. Sci. 88, 11120-11123).
[0066] Antibodies can also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as is well-known in the art (Orlandi, et al. (1989) Proc.
Natl. Acad. Sci. 86: 3833-3837; Winter, et al. (1991) Nature
349:293-299).
[0067] Diabodies are also contemplated. A diabody refers to an
engineered antibody construct prepared by isolating the binding
domains (both heavy and light chain) of a binding antibody, and
supplying a linking moiety which joins or operably links the heavy
and light chains on the same polypeptide chain thereby preserving
the binding function (see, Holliger et al. (1993) Proc. Natl. Acad.
Sci. USA 90:6444; Poljak (1994) Structure 2:1121-1123). This forms,
in essence, a radically abbreviated antibody, having only the
variable domain necessary for binding the antigen. By using a
linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the
complementary domains of another chain and create two
antigen-binding sites. These dimeric antibody fragments, or
diabodies, are bivalent and bispecific. It should be clear that any
method to generate diabodies, as for example described by Holliger,
et al. (1993) supra, Poljak (1994) supra, Zhu, et al. (1996)
Biotechnology 14:192-196, and U.S. Pat. No. 6,492,123, herein
incorporated by reference, can be used. In one embodiment, an
antibody or diabody of the present invention is a bispecific
agonistic antibody which specifically agonizes CD3 and
PD-L3-receptors and co-stimulates T cell activation.
[0068] Various immunoassays can be used for screening to identify
antibodies, or fragments thereof, having the desired specificity
for PD-L3 protein. Numerous protocols for competitive binding (e.g,
ELISA), latex agglutination assays, immunoradiometric assays, and
kinetics (e.g. BIACORE.TM. analysis) using either polyclonal or
monoclonal antibodies, or fragments thereof, and are well-known in
the art. Such immunoassays typically involve the measurement of
complex formation between a specific antibody and its cognate
antigen. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes is
suitable, but a competitive binding assay can also be employed.
[0069] An antibody of the instant invention is useful producing a
corresponding anti-idiotypic antibody. Anti-idiotypic antibodies,
or anti-idiotypes are antibodies directed against the
antigen-combining region or variable region (idiotype) of another
antibody. Based on Jerne's network model of idiotypic relationships
(Jerne (1974) Ann. Immunol. 125:373; Jerne, et al. (1982) EMBO J.
1:234), immunization with an antibody molecule expressing a
paratope (antigen-combining site) for a given antigen produces a
group of anti-antibodies, some of which share with the antigen a
complementary structure to the paratope. Such anti-idiotypic
antibodies would be useful for antagonizing or agonizing the PD-L3
receptor.
[0070] In one embodiment, an anti-PD-L3 antibody, or peptide
aptamer, of the instant invention is agonistic which, like PD-L3
protein, binds a PD-L3 receptor and activates the receptor. In
another embodiment, the anti-PD-L3 antibody, or peptide aptamer, is
antagonistic and blocks the binding of PD-L3 protein to its cognate
receptor on an immune cell thereby blocking activation of the
receptor. Like a PD-L3 protein, such PD-L3 binding agents are
useful in methods for modulating an immune cell response.
[0071] Also encompassed by the present invention are small
molecules which can modulate (either enhance or inhibit)
interactions between PD-L3 and its cognate receptor(s). Such small
molecules can be obtained using any of the numerous approaches in
combinatorial library methods known in the art, including spatially
addressable parallel solid phase or solution phase libraries;
synthetic library methods requiring deconvolution; the one-bead
one-compound library method; and synthetic library methods using
affinity chromatography selection. (Lam (1997) Anticancer Drug Des.
12:145).
[0072] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in, DeWitt, et al. (1993)
Proc. Natl. Acad. Sci. USA 90:6909; Erb, et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann, et al. (1994) J. Med. Chem.
37:2678; Cho, et al. (1993) Science 261:1303; Carrell, et al.
(1994) Angew. Chem. Int. Ed. Engl. 33:2059; and Gallop, et al.
(1994) J. Med. Chem. 37:1233.
[0073] Libraries of compounds can be presented in solution (e.g.,
Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria or spores (U.S. Pat. No. 5,223,409), plasmids (Cull, et
al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage
(Scott and Smith (1990) Science 249:386-390; Devlin (1990) Science
249:404-406; Cwirla, et al. (1990) Proc. Natl. Acad. Sci. USA
87:6378-6382; Felici (1991) J. Mol. Biol. 222:301-310). Compounds
can be screened in cell-based or non-cell-based assays. Compounds
can be screened in pools (e.g., multiple compounds in each testing
sample) or as individual compounds.
[0074] A small molecule for modulating interactions between PD-L3
and its cognate receptor can be identified, for example, in a
cell-based assay. Such an assay involves contacting a cell
expressing a PD-L3 receptor (e.g., a T cell), with a test molecule
and determining the ability of the test molecule to modulate (e.g.,
stimulate or inhibit) the binding of PD-L3 to its binding partner.
Determining the ability of the PD-L3 to bind to, or interact with,
its binding partner can be accomplished, e.g., by measuring direct
binding or by measuring a parameter of immune cell activation
(e.g., cell proliferation or cytokine production). In a direct
binding assay, the PD-L3 protein can be coupled with a radioisotope
(e.g., .sup.125I, .sup.35S, .sup.14C, or .sup.3H) such that binding
of PD-L3 can be determined by detecting the labeled protein in a
complex (e.g., direct counting of radioemmission or by
scintillation counting). Alternatively, PD-L3 can be enzymatically
labeled with, for example, horseradish peroxidase, alkaline
phosphatase, or luciferase, and the enzymatic label detected by
determination of conversion of an appropriate substrate to
product.
[0075] In one embodiment, a small molecule binds to antagonizes the
interaction between PD-L3 and at least one cognate receptor. in
another embodiment, the small molecule agonizes the interaction
between PD-L3 and at least one cognate receptor.
[0076] PD-L3 protein, PD-L3 binding agents (antagnostic or
agonistic), small molecule agonists or antagonists, vectors,
isolated host cells or T cells activated by the methods described
herein can be used for modulating immune responses in subjects for
treating and preventing cancer, infectious disease, autoimmune
disease, immune dysfunction related to aging, transplantation or
any other disease state where such agents are desired for
treatment. Such modulatory agents (i.e., PD-L3 protein, PD-L3
binding agents, small molecule agonists or antagonists, vectors,
isolated host cells or activated T cells) can be administered
either alone, or as a pharmaceutical composition in combination
with diluents and/or with other components such as cytokines or
other cell populations (e.g., APCs). In general, a pharmaceutical
composition containing a modulatory agent is formulated with one or
more pharmaceutically acceptable carriers such as those described
herein.
[0077] A modulatory agent described herein can be used in methods
of prevention or treatment (e.g., by up- or down-modulating the
immune response). Such methods involve administration to a subject,
at risk of having or having a disease or condition associated with
an unwanted or less than desirable immune response, a prophylactic
or therapeutic agent prior to or after the manifestation of
symptoms associated with an unwanted or less than desirable immune
response. Subjects at risk or having a disease that would benefit
from treatment with such agents or methods can be identified, for
example, by any or a combination of diagnostic or prognostic assays
known in the art. The appropriate agent used for treatment (e.g.
antibodies, peptides, fusion proteins or small molecules) can be
determined based on clinical indications and can be identified,
e.g., using screening assays described herein. Modulatory agents
can be administered in vitro (e.g., by contacting the cell with the
agent) or, alternatively, in vivo (e.g., by administering the agent
to a subject). As such, the present invention relates to methods of
treating an individual afflicted with a disease or disorder that
would benefit from modulation of an immune response, e.g., by
modulation of the interaction between a PD-L3 ligand its cognate
receptor(s). As PD-L3 expression was predominantly localized to the
brain, one embodiment of the present invention is the prevention or
treatment of a disease or condition of the brain.
[0078] To illustrate the efficacy of using a PD-L3 protein,
co-stimulation (i.e., CD3/CD30-mediated) has been successfully used
in in vivo tumor-specific activation of the T cell cytolytic
machinery (see, Bauer, et al. (1999) Cancer Res. 59:1961-5).
Likewise, CD3/CD28-activated T cells and interleukin-2
administration has achieved tumor regression in bone metastases in
Phase I clinical trials of metastatic renal cell carcinoma
(Thompson, et al. (Clin. Cancer Res. 2003 Sep. 1; 9 (10 Pt
1):3562-7). Accordingly, it is contemplated that co-stimulation
with PD-L3 in combination with a primary signal (e.g., anti-CD3)
will be useful for modulating cellular immune responses mediated by
cytotoxic T cells, capable of killing tumor and infected cells, and
helper T cell responses.
[0079] The invention is described in greater detail by the
following non-limiting examples.
Example 1
Expression Profiling
[0080] To facilitate comparisons with established expression
profiles of T.sup.reg cells, standard growth and activation
conditions were employed (McHugh, et al. (2002) supra). Briefly,
fresh isolated T.sup.reg cells (.about.96% positive) were
inoculated at 10.sup.6/mL into complete RPMI medium supplemented
with 10% fetal bovine serum and 100 units IL-2 in a 24-well plate
precoated with anti-CD3 with or without anti-GITR (DTA-1) (Shimizu,
et al. (2002) supra). The cells were cultured at 37.degree. C. for
0 and 12 hours, RNA was purified and subsequently analyzed using an
AFFYMETRIX.RTM. mouse genome A430 oligonucleotide array.
[0081] By comparing the data from resting or activated
CD4.sup.+CD25.sup.+ T cell groups, gene expression patterns were
found to be similar to those established in the art (Gavin, et al.
(2002) supra; McHugh, et al. (2002) supra). It identify genes
regulated by GIRT signaling, gene expression profiles were compared
between the different cell populations with or without anti-GITR
treatment. A list of known as well as unknown genes were compiled
including the previously uncharacterized PD-L3 and
T.sup.reg-sTNF.
Example 2
Inhibitory Activity of PD-L3
[0082] The inhibitory activity of PD-L1 was revealed by using
antigen presenting cells over-expressing PD-L1 in vitro with
CD4.sup.+ and CD8.sup.+ T cell antigen receptor transgenic T cells
and antigen stimulation (Carter, et al. (2002) Eur. J. Immunol.
32:634-43). Similarly, the lentivector disclosed herein, which
expresses the full-length PD-L3, is transduced into cell lines
expressing class II major histocompatibility complex (MHC) and
class I MHC. The response of TEa Tg or the 2C transgenic T cells to
antigen presented by empty vector-transduced or PD-L3-transduced
antigen presenting cells is determined according to established
methods.
Example 3
Protein Expression
[0083] Expression patterns in lymphoid, monocyte and dendritic cell
subsets, as well as non-hemoatopoietic tissues, is determined by
RT-PCR and western blot analysis using standard protocols in
combination with the rabbit .alpha.PD-L3 antibody disclosed
herein.
Example 4
PD-L3 Transgenic Mice
[0084] Using Lentiviral infection of embryos, four transgenic mice
ubiquitously expressing PD-L3 have been produced. These mice are
expected to spontaneously develop autoimmunity and in vivo immune
responses in the PD-L3 transgenic mice (i.e., humoral immune
responses, T cell priming, etc.) are evaluated to assess systemic
autoimmune disease development.
Example 5
PD-L3 Knock-Out Mice
[0085] PD-L3 is inactivated by homologous recombination. A BAC
clone containing full-length PD-L3 sequence was purchased from
INVITROGEN.TM. (Carlsbad, Calif.). A PD-L3 targeting vector was
generated by inserting a 1.6 kb fragment located at the 5' side of
the second exon of PD-L3 gene upstream the neomycin gene and the 5
kb fragment located at the 3.degree. side of the third exon of
PD-L3 gene downstream the neomycin gene. B6-derived embryonic stem
(ES) cells are electroporated with PD-L3 targeting vector and
recombined clones are selected. Selected clones are then injected
into C57BL/6 blastocytes and the resulting chimeric male offspring
are mated to FLP-deleter mice to remove the neomycin cassette.
Transmission of the targeted allele in the offspring is determined
by PCR from genomic DNA. The second and the third exon contain the
PD-L3 domain, therefore, the resulting mice have only the
inactivated form of the PD-L3 molecule.
[0086] The overall immune capacity of PD-L3 deficient mice is
determined as with other PD-L.sup.-/- mice, including assessment of
T cell responses to antigen, humoral immune responses, overt
autoimmunity (Systemic Lupus Erythematosus, inflammatory bowel
disease), and increased susceptibility to induced autoimmune
disease (experimental autoimmune encephalomyelitis) (Chen (2004)
supra).
Sequence CWU 1
1
1014795DNAMus musculus 1gagcattcac tctagcgagc gagcggcgtg tacagccggc
tccctgggct cctggagtcc 60cgcttgctcc aagcgcactc cagcagtctc tttctgctct
tgcccggctc gacggcgaca 120tgggtgtccc cgcggtccca gaggccagca
gcccgcgctg gggaaccctg ctccttgcta 180ttttcctggc tgcatccaga
ggtctggtag cagccttcaa ggtcaccact ccatattctc 240tctatgtgtg
tcccgaggga cagaatgcca ccctcacctg caggattctg ggccccgtgt
300ccaaagggca cgatgtgacc atctacaaga cgtggtacct cagctcacga
ggcgaggtcc 360agatgtgcaa agaacaccgg cccatacgca acttcacatt
gcagcacctt cagcaccacg 420gaagccacct gaaagccaac gccagccatg
accagcccca gaagcatggg ctagagctag 480cttctgacca ccacggtaac
ttctctatca ccctgcgcaa tgtgacccca agggacagcg 540gcctctactg
ctgtctagtg atagaattaa aaaaccacca cccagaacaa cggttctacg
600ggtccatgga gctacaggta caggcaggca aaggctcggg gtccacatgc
atggcgtcta 660atgagcagga cagtgacagc atcacggctg cggccctggc
caccggcgcc tgcatcgtgg 720gaatcctctg cctccccctt atcctgctgc
tggtctataa gcagagacag gtggcctctc 780accgccgtgc ccaggagttg
gtgaggatgg acagcagcaa cacccaagga atcgaaaacc 840caggcttcga
gaccactcca cccttccagg ggatgcctga ggccaagacc aggccgccac
900tgtcctatgt ggcccagcgg caaccttcgg agtcaggacg gtacctgctc
tctgacccca 960gcacacctct gtcgcctcca ggccctgggg acgtcttttt
cccatcccta gatccagtcc 1020ctgactcccc taactctgaa gccatctaaa
ccagctgggg aaccatgaac catggtacct 1080gggtcaggga tatgtgcact
tgatctatgg ctggcccttg gacagtcttt taggcactga 1140ctccagcttc
cttgctcctg ctctgagcct agactctgct tttacaagat gcacagaccc
1200tcccctatct ctttcagacg ctacttgggg ggcagggaga agatgttgga
ttgctcattg 1260ctgttctcaa gatcttggga tgctgagttc tccctagaga
cttgacttcg acagccacag 1320atgtcagatg acctgcatcc tatgaacgtc
cggcttggca agagcctttc ttcatggaaa 1380ccagtagccc ggaggggatg
aggtaggcac cttgccaccc tcccgggaga gagacacaag 1440atgtgagaga
ctcctgctca ctgtgggggt gtggctggcc tgcttgtttg cctgaggatg
1500ctcctctgtt ggactgactc tatccccctg gattctggag cttggctggc
ctatgtccca 1560ccagaggagc atctcagcag ccttccacca gcaacctgag
ggcctgccag cttcgtggct 1620ctgggctctc attacctgta tggccgtcca
cagagctcag tggccagagg ctttgaaaca 1680ggaagtacat gtcaggttca
ggaaccactg tgagctcatt agtgtcttga gcaatgtgag 1740gcctggacca
gtggacacgg agggagggtg gcgagaggat gatggggatg atgaggggaa
1800cacgctccct tcctgtcctt gtcatccacc actaccacta ttcagtgtgg
agcagtggca 1860aaggtgaccg acctccacaa tgtcctagtg atgctggacc
atttctaagt gtgaaagaga 1920tgctattaaa aacagtatgt ggcaatggct
gccaacagct gagtggactg gaggcactgg 1980ctttaaggcc ctggaggtgc
agggcccggt atggggatag ggatgggagt ttcagtgagg 2040gcctagggat
cactccgctt ctgaccactc ttcttctgag cctcacctca gggtgacctt
2100caggcacaca gaagagcttg cccctggtcc gatactactc ttggctctca
tctccagggt 2160ttggcatgac ctgggcacac agggggagtc ttcagaaagg
attttaaagc atgaaaagaa 2220agggtagttc ttgtgaggta gggatgggca
gctgatgttt gagagtgagg agggatacgg 2280ctgggcagat cactctccag
tctctagagg gaaagtagct ctaagtctgg gagagcagca 2340gcccagtggt
accatatgtc ttcttgcagc ttccactggc tgggctgaac tgggcatggg
2400taggaaagct cctgttctgg gcctgcagcc agggagaacc ccattcattc
cctgaggaca 2460gatgggtggg gagagaagag agagtttcag gccgggaagc
agcaataagc tatctgctgg 2520ggacccagac aagttgtctg atgaggtcca
agatgtggga tgccagttat acctggggct 2580tggggatcct tagaggcttt
gtatcatcat cataggagtg tcggggtggc cagggcatca 2640aagccatgac
ccctgtttta tcctcagggt ccactcttct gcaccatcca ttgctctaga
2700tctatgcagt tactatagac agaatgtgtt gttctgtttg gctttgggga
taatggcctg 2760gcgaactgcc agctgttcag tggcagggct gtgaggccag
tcaaagacta gaacccacag 2820accagctgaa cgatgagtat agcctgtccc
ctgggggagc ctgacctgtc tccagcccta 2880agcttcagac ctcaccactc
agatgacttc taagaatttg cctgtgggga cccctgcatg 2940gctgcagctc
cgtggaaagg agaggaggcc cccagcagaa gaaccactcg cttcctgccc
3000agcttcctcc tgtagggctc taagtctctt cttcttggga ccctgcaagc
aaaggcatgt 3060cagcttggtg gtttcctgtt ttgggtgaag ttttgtgtgg
tccgggttct gtctacatcc 3120atgaacttgg ggtgctacca ccttgctgct
gctgtagaga cagctgcagg atcttagggt 3180ggaaaatgga ggtgccctga
ggtgctagcc cttggggcaa aagatggggt ggcaatgaga 3240cacagtgggg
aactgagttc cccaagagga gggaggagcc ctgtagcctc aagggccata
3300ttgggttcct ggtaccagca aaagcctaga gagcgaagtc tgtattttga
ggaggtaatt 3360gatccttacg gaatccatca gaaatttgga gcgggtgctt
tatctatctc tggagggtct 3420ctacctatct ccgatgaagc tctccctggg
cctgggatgg gagaaaccag gaggaaaggt 3480gtctgataaa gcaggggctt
cttgacaagc caaagggcca ctggtagctg ttgtggaccg 3540agctgaccct
gctgaagtat tgtagtgtgc cttggaccaa cttctcaaaa gagcaacccc
3600ggggctaccc tacttctgcc aggaagaggc ggagaagggg ctgagaggcc
tggaaggggc 3660tagctccttc tttgagaact gctccccgga ggacttggag
gaggcggcta ggctacgggc 3720tgctgagggc cctttgtctt tcctaacctg
ggcactgtta ggatgctccc tcctggaaaa 3780ggctttcctg ggtgtgagct
agagcagtgt ccatgccagc gctgaacctg ccatggtggg 3840agctgaacta
aaaatttctc agggaactaa aataggcaaa agaggaactg ggggaggagg
3900gtgccaggca ggatgggggg aagggagggc agtgcaaaag tctcttgaaa
cacagacagc 3960ccagctgagt gccagtccca gatcacagag aatacggctc
atctggctca tgttctgcat 4020gcttgctgct ttaccctggc actttccttc
tccaccatga gtgcgagtcc tgggagtcct 4080gggagggtga ggattaatgc
cagcctgggg agcagatagc tgacagagtc cttgggtaac 4140tggcttgaac
caggacctca ggattccact ctggggatct agctttgtct gggccagtga
4200agatctctat aatggcatta ttgccagggg ataaacattt cactgggttc
tgatctgttg 4260ggtgtggctt cctggaaaat atggtgagag gaattctgct
aaggatacag ttgataagaa 4320agttctgaga ttgattagta atgcctgcct
tggactcagg aagggaagtg gcagtatgaa 4380tgccatgtct taatcatttt
ggttaaaata tgcttcccaa aagatttcca cgtgtgttct 4440tgtttatttg
acatctgtct ccatatcagt cttgaaagcc tttctgtgtg tatatatatg
4500atgtttgcgt gtatatatgt ttttgtgtgt gcatatggaa gtcagaaatc
actgggtgtc 4560ttcctccatt cctttgcaat gtatgttttt ttttttttta
cgatttattt actatatgaa 4620tgttttgcct gaatacatgc ataggtgtca
cgtacatgcc tgctggaacg cttggaactg 4680gagttacagg tggctatgag
ctacagtgtg agcactggga atcaaacctg ggtcttctgc 4740aagagcaaca
aattaaaagt cagctcttaa ctacttgagc tatttttcca actcc 47952309PRTMus
musculus 2Met Gly Val Pro Ala Val Pro Glu Ala Ser Ser Pro Arg Trp
Gly Thr1 5 10 15Leu Leu Leu Ala Ile Phe Leu Ala Ala Ser Arg Gly Leu
Val Ala Ala 20 25 30Phe Lys Val Thr Thr Pro Tyr Ser Leu Tyr Val Cys
Pro Glu Gly Gln 35 40 45Asn Ala Thr Leu Thr Cys Arg Ile Leu Gly Pro
Val Ser Lys Gly His 50 55 60Asp Val Thr Ile Tyr Lys Thr Trp Tyr Leu
Ser Ser Arg Gly Glu Val65 70 75 80Gln Met Cys Lys Glu His Arg Pro
Ile Arg Asn Phe Thr Leu Gln His 85 90 95Leu Gln His His Gly Ser His
Leu Lys Ala Asn Ala Ser His Asp Gln 100 105 110Pro Gln Lys His Gly
Leu Glu Leu Ala Ser Asp His His Gly Asn Phe 115 120 125Ser Ile Thr
Leu Arg Asn Val Thr Pro Arg Asp Ser Gly Leu Tyr Cys 130 135 140Cys
Leu Val Ile Glu Leu Lys Asn His His Pro Glu Gln Arg Phe Tyr145 150
155 160Gly Ser Met Glu Leu Gln Val Gln Ala Gly Lys Gly Ser Gly Ser
Thr 165 170 175Cys Met Ala Ser Asn Glu Gln Asp Ser Asp Ser Ile Thr
Ala Ala Ala 180 185 190Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu
Cys Leu Pro Leu Ile 195 200 205Leu Leu Leu Val Tyr Lys Gln Arg Gln
Val Ala Ser His Arg Arg Ala 210 215 220Gln Glu Leu Val Arg Met Asp
Ser Ser Asn Thr Gln Gly Ile Glu Asn225 230 235 240Pro Gly Phe Glu
Thr Thr Pro Pro Phe Gln Gly Met Pro Glu Ala Lys 245 250 255Thr Arg
Pro Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser Glu Ser 260 265
270Gly Arg Tyr Leu Leu Ser Asp Pro Ser Thr Pro Leu Ser Pro Pro Gly
275 280 285Pro Gly Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp
Ser Pro 290 295 300Asn Ser Glu Ala Ile30534774DNAHomo sapiens
3gggggcgggt gcctggagca cggcgctggg gccgcccgca gcgctcactc gctcgcactc
60agtcgcggga ggcttccccg cgccggccgc gtcccgcccg ctccccggca ccagaagttc
120ctctgcgcgt ccgacggcga catgggcgtc cccacggccc tggaggccgg
cagctggcgc 180tggggatccc tgctcttcgc tctcttcctg gctgcgtccc
taggtccggt ggcagccttc 240aaggtcgcca cgccgtattc cctgtatgtc
tgtcccgagg ggcagaacgt caccctcacc 300tgcaggctct tgggccctgt
ggacaaaggg cacgatgtga ccttctacaa gacgtggtac 360cgcagctcga
ggggcgaggt gcagacctgc tcagagcgcc ggcccatccg caacctcacg
420ttccaggacc ttcacctgca ccatggaggc caccaggctg ccaacaccag
ccacgacctg 480gctcagcgcc acgggctgga gtcggcctcc gaccaccatg
gcaacttctc catcaccatg 540cgcaacctga ccctgctgga tagcggcctc
tactgctgcc tggtggtgga gatcaggcac 600caccactcgg agcacagggt
ccatggtgcc atggagctgc aggtgcagac aggcaaagat 660gcaccatcca
actgtgtggt gtacccatcc tcctcccagg atagtgaaaa catcacggct
720gcagccctgg ctacgggtgc ctgcatcgta ggaatcctct gcctccccct
catcctgctc 780ctggtctaca agcaaaggca ggcagcctcc aaccgccgtg
cccaggagct ggtgcggatg 840gacagcaaca ttcaagggat tgaaaacccc
ggctttgaag cctcaccacc tgcccagggg 900atacccgagg ccaaagtcag
gcaccccctg tcctatgtgg cccagcggca gccttctgag 960tctgggcggc
atctgctttc ggagcccagc acccccctgt ctcctccagg ccccggagac
1020gtcttcttcc catccctgga ccctgtccct gactctccaa actttgaggt
catctagccc 1080agctggggga cagtgggctg ttgtggctgg gtctggggca
ggtgcatttg agccagggct 1140ggctctgtga gtggcctcct tggcctcggc
cctggttccc tccctcctgc tctgggctca 1200gatactgtga catcccagaa
gcccagcccc tcaacccctc tggatgctac atggggatgc 1260tggacggctc
agcccctgtt ccaaggattt tggggtgctg agattctccc ctagagacct
1320gaaattcacc agctacagat gccaaatgac ttacatctta agaagtctca
gaacgtccag 1380cccttcagca gctctcgttc tgagacatga gccttgggat
gtggcagcat cagtgggaca 1440agatggacac tgggccaccc tcccaggcac
cagacacagg gcacggtgga gagacttctc 1500ccccgtggcc gccttggctc
ccccgttttg cccgaggctg ctcttctgtc agacttcctc 1560tttgtaccac
agtggctctg gggccaggcc tgcctgccca ctggccatcg ccaccttccc
1620cagctgcctc ctaccagcag tttctctgaa gatctgtcaa caggttaagt
caatctgggg 1680cttccactgc ctgcattcca gtccccagag cttggtggtc
ccgaaacggg aagtacatat 1740tggggcatgg tggcctccgt gagcaaatgg
tgtcttgggc aatctgaggc caggacagat 1800gttgccccac ccactggaga
tggtgctgag ggaggtgggt ggggccttct gggaaggtga 1860gtggagaggg
gcacctgccc cccgccctcc ccatccccta ctcccactgc tcagcgcggg
1920ccattgcaag ggtgccacac aatgtcttgt ccaccctggg acacttctga
gtatgaagcg 1980ggatgctatt aaaaactaca tggggaaaca ggtgcaaacc
ctggagatgg attgtaagag 2040ccagtttaaa tctgcactct gctgctcctc
ccccaccccc accttccact ccatacaatc 2100tgggcctggt ggagtcttcg
cttcagagcc attcggccag gtgcgggtga tgttcccatc 2160tcctgcttgt
gggcatgccc tggctttgtt tttatacaca taggcaaggt gagtcctctg
2220tggaattgtg attgaaggat tttaaagcag gggaggagag tagggggcat
ctctgtacac 2280tctgggggta aaacagggaa ggcagtgcct gagcatgggg
acaggtgagg tggggctggg 2340cagaccccct gtagcgttta gcaggatggg
ggccccaggt actgtggaga gcatagtcca 2400gcctgggcat ttgtctccta
gcagcctaca ctggctctgc tgagctgggc ctgggtgctg 2460aaagccagga
tttggggcta ggcgggaaga tgttcgccca attgcttggg gggttggggg
2520gatggaaaag gggagcacct ctaggctgcc tggcagcagt gagccctggg
cctgtggcta 2580cagccaggga accccacctg gacacatggc cctgcttcta
agccccccag ttaggcccaa 2640aggaatggtc cactgagggc ctcctgctct
gcctgggctg ggccaggggc tttgaggaga 2700gggtaaacat aggcccggag
atggggctga cacctcgagt ggccagaata tgcccaaacc 2760ccggcttctc
ccttgtccct aggcagaggg gggtcccttc ttttgttccc tctggtcacc
2820acaatgcttg atgccagctg ccataggaag agggtgctgg ctggccatgg
tggcacacac 2880ctgtcctccc agcactttgc agggctgagg tggaaggacc
gcttaagccc aggtgttcaa 2940ggctgctgtg agctgtgttc gagccactac
actccagcct ggggacggag caaaactttg 3000cctcaaaaca aattttaaaa
agaaagaaag aaggaaagag ggtatgtttt tcacaattca 3060tgggggcctg
catggcagga gtggggacag gacacctgct gttcctggag tcgaaggaca
3120agcccacagc ccagattccg gttctcccaa ctcaggaaga gcatgccctg
ccctctgggg 3180aggctggcct ggccccagcc ctcagctgct gaccttgagg
cagagacaac ttctaagaat 3240ttggctgcca gaccccaggc ctggctgctg
ctgtgtggag agggaggcgg cccgcagcag 3300aacagccacc gcacttcctc
ctcagcttcc tctggtgcgg ccctgccctc tcttctctgg 3360acccttttac
aactgaacgc atctgggctt cgtggtttcc tgttttcagc gaaatttact
3420ctgagctccc agttccatct tcatccatgg ccacaggccc tgcctacaac
gcactaggga 3480cgtccctccc tgctgctgct ggggaggggc aggctgctgg
agccgccctc tgagttgccc 3540gggatggtag tgcctctgat gccagccctg
gtggctgtgg gctggggtgc atgggagagc 3600tgggtgcgag aacatggcgc
ctccaggggg cgggaggagc actaggggct ggggcaggag 3660gctcctggag
cgctggattc gtggcacagt ctgaggccct gagagggaaa tccatgcttt
3720taagaactaa ttcattgtta ggagatcaat caggaattag gggccatctt
acctatctcc 3780tgacattcac agtttaatag agacttcctg cctttattcc
ctcccaggga gaggctgaag 3840gaatggaatt gaaagcacca tttggagggt
tttgctgaca cagcggggac tgctcagcac 3900tccctaaaaa cacaccatgg
aggccactgg tgactgctgg tgggcaggct ggccctgcct 3960gggggagtcc
gtggcgatgg gcgctggggt ggaggtgcag gagccccagg acctgctttt
4020caaaagactt ctgcctgacc agagctccca ctacatgcag tggcccaggg
cagaggggct 4080gatacatggc ctttttcagg gggtgctcct cgcggggtgg
acttgggagt gtgcagtggg 4140acagggggct gcaggggtcc tgccaccacc
gagcaccaac ttggcccctg gggtcctgcc 4200tcatgaatga ggccttcccc
agggctggcc tgactgtgct gggggctggg ttaacgtttt 4260ctcagggaac
cacaatgcac gaaagaggaa ctggggttgc taaccaggat gctgggaaca
4320aaggcctctt gaagcccagc cacagcccag ctgagcatga ggcccagccc
atagacggca 4380caggccacct ggcccattcc ctgggcattc cctgctttgc
attgctgctt ctcttcaccc 4440catggaggct atgtcaccct aactatcctg
gaatgtgttg agagggattc tgaatgatca 4500atatagcttg gtgagacagt
gccgagatag atagccatgt ctgccttggg cacgggagag 4560ggaagtggca
gcatgcatgc tgtttcttgg ccttttctgt tagaatactt ggtgctttcc
4620aacacacttt cacatgtgtt gtaacttgtt tgatccaccc ccttccctga
aaatcctggg 4680aggttttatt gctgccattt aacacagagg gcaatagagg
ttctgaaagg tctgtgtctt 4740gtcaaaacaa gtaaacggtg gaactacgac taaa
47744311PRTHomo sapiens 4Met Gly Val Pro Thr Ala Leu Glu Ala Gly
Ser Trp Arg Trp Gly Ser1 5 10 15Leu Leu Phe Ala Leu Phe Leu Ala Ala
Ser Leu Gly Pro Val Ala Ala 20 25 30Phe Lys Val Ala Thr Pro Tyr Ser
Leu Tyr Val Cys Pro Glu Gly Gln 35 40 45Asn Val Thr Leu Thr Cys Arg
Leu Leu Gly Pro Val Asp Lys Gly His 50 55 60Asp Val Thr Phe Tyr Lys
Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val65 70 75 80Gln Thr Cys Ser
Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp 85 90 95Leu His Leu
His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp 100 105 110Leu
Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn 115 120
125Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu Tyr
130 135 140Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu His
Arg Val145 150 155 160His Gly Ala Met Glu Leu Gln Val Gln Thr Gly
Lys Asp Ala Pro Ser 165 170 175Asn Cys Val Val Tyr Pro Ser Ser Ser
Gln Asp Ser Glu Asn Ile Thr 180 185 190Ala Ala Ala Leu Ala Thr Gly
Ala Cys Ile Val Gly Ile Leu Cys Leu 195 200 205Pro Leu Ile Leu Leu
Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn 210 215 220Arg Arg Ala
Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln Gly Ile225 230 235
240Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala Gln Gly Ile Pro Glu
245 250 255Ala Lys Val Arg His Pro Leu Ser Tyr Val Ala Gln Arg Gln
Pro Ser 260 265 270Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser Thr
Pro Leu Ser Pro 275 280 285Pro Gly Pro Gly Asp Val Phe Phe Pro Ser
Leu Asp Pro Val Pro Asp 290 295 300Ser Pro Asn Phe Glu Val Ile305
310530PRTArtificial SequenceConsensus sequence for PD-L3 5Ile Thr
Ala Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu1 5 10 15Cys
Leu Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln 20 25
3061316DNAMus musculus 6ggagtcctcc ccttggagcc tgggaggcct agggagaaag
tagttctctt tcggtggcag 60ggttgctgtc gagggcaccg agcaggagga taggtcgaca
gagacgagga gttctggctc 120ctcctgcaga catgcaccag cggctgctgg
gctcgtccct gggcctcgcc cccgcgcggg 180ggctctgaat gcctgccgcc
gcccccatga gagcaccggc ctgggctccc gcccctaagc 240ctctgctcgc
ggagactgag ccatgtgggc ctggggctgg gccgctgcag cgctcctctg
300gctacagact gcaggagccg gggcccggca ggagctcaag aagtctcggc
agctgtttgc 360gcgtgtggat tcccccaata ttaccacgtc caaccgtgag
ggattcccag gctccgtcaa 420gcccccggaa gcctctggac ctgagctctc
agatgcccac atgacgtggt tgaactttgt 480ccgacggcca gatgatgggt
cctctagaaa acggtgtcgt ggccgggaca agaagtcgcg 540aggcctctca
ggtctcccag ggcccccagg acctcctggc cctcctggtc cccctggctc
600ccctggtgtg ggcgttaccc cagaggcctt actgcaggaa tttcaggaga
tactgaaaga 660ggccacagaa cttcgattct cagggctacc agacacattg
ttaccccagg aacccagcca 720acggctggtg gttgaggcct tctactgccg
tttgaaaggc cctgtgctgg tggacaagaa 780gactctggtg gaactgcaag
gattccaagc tcctactact cagggcgcct tcctgcgggg 840atctggcctg
agcctgtcct tgggccgatt cacagcccca gtctctgcca tcttccagtt
900ttctgccagc ctgcacgtgg accacagtga actgcagggc agaggccggt
tgcgtacccg 960ggatatggtc cgtgttctca tctgtattga gtccttgtgt
catcgtcata cgtccctgga 1020ggctgtatca ggtctggaga gcaacagcag
ggtcttcaca gtgcaggttc aggggctgct 1080gcatctacag tctggacagt
atgtctctgt gttcgtggac aacagttctg gggcagtcct 1140caccatccag
aacacttcca gcttctcggg aatgcttttg ggtacctagc ggagctgaag
1200aaacgattgt ggattgagga accaacacct tgcttcttag aggagctgaa
aaggactact 1260cactcccctt ttaatagttt tcatagcaat aaagaactcc
aaacttcttc atcgct 13167308PRTMus musculus 7Met Trp Ala Trp Gly Trp
Ala Ala Ala Ala Leu Leu
Trp Leu Gln Thr1 5 10 15Ala Gly Ala Gly Ala Arg Gln Glu Leu Lys Lys
Ser Arg Gln Leu Phe 20 25 30Ala Arg Val Asp Ser Pro Asn Ile Thr Thr
Ser Asn Arg Glu Gly Phe 35 40 45Pro Gly Ser Val Lys Pro Pro Glu Ala
Ser Gly Pro Glu Leu Ser Asp 50 55 60Ala His Met Thr Trp Leu Asn Phe
Val Arg Arg Pro Asp Asp Gly Ser65 70 75 80Ser Arg Lys Arg Cys Arg
Gly Arg Asp Lys Lys Ser Arg Gly Leu Ser 85 90 95Gly Leu Pro Gly Pro
Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 100 105 110Ser Pro Gly
Val Gly Val Thr Pro Glu Ala Leu Leu Gln Glu Phe Gln 115 120 125Glu
Ile Leu Lys Glu Ala Thr Glu Leu Arg Phe Ser Gly Leu Pro Asp 130 135
140Thr Leu Leu Pro Gln Glu Pro Ser Gln Arg Leu Val Val Glu Ala
Phe145 150 155 160Tyr Cys Arg Leu Lys Gly Pro Val Leu Val Asp Lys
Lys Thr Leu Val 165 170 175Glu Leu Gln Gly Phe Gln Ala Pro Thr Thr
Gln Gly Ala Phe Leu Arg 180 185 190Gly Ser Gly Leu Ser Leu Ser Leu
Gly Arg Phe Thr Ala Pro Val Ser 195 200 205Ala Ile Phe Gln Phe Ser
Ala Ser Leu His Val Asp His Ser Glu Leu 210 215 220Gln Gly Arg Gly
Arg Leu Arg Thr Arg Asp Met Val Arg Val Leu Ile225 230 235 240Cys
Ile Glu Ser Leu Cys His Arg His Thr Ser Leu Glu Ala Val Ser 245 250
255Gly Leu Glu Ser Asn Ser Arg Val Phe Thr Val Gln Val Gln Gly Leu
260 265 270Leu His Leu Gln Ser Gly Gln Tyr Val Ser Val Phe Val Asp
Asn Ser 275 280 285Ser Gly Ala Val Leu Thr Ile Gln Asn Thr Ser Ser
Phe Ser Gly Met 290 295 300Leu Leu Gly Thr30581055DNAHomo sapiens
8ctcgccgcgc tgagccgcct cgggacggag ccatgcggcg ctgggcctgg gccgcggtcg
60tggtcctcct cgggccgcag ctcgtgctcc tcgggggcgt cggggcccgg cgggaggcac
120agaggacgca gcagcctggc cagcgcgcag atccccccaa cgccaccgcc
agcgcgtcct 180cccgcgaggg gctgcccgag gcccccaagc catcccaggc
ctcaggacct gagttctccg 240acgcccacat gacatggctg aactttgtcc
ggcggccgga cgacggcgcc ttaaggaagc 300ggtgcggaag cagggacaag
aagccgcggg atctcttcgg tcccccagga cctccaggtg 360cagaagtgac
cgcggagact ctgcttcacg agtttcagga gctgctgaaa gaggccacgg
420agcgccggtt ctcagggctt ctggacccgc tgctgcccca gggggcgggc
ctgcggctgg 480tgggcgaggc ctttcactgc cggctgcagg gtccccgccg
ggtggacaag cggacgctgg 540tggagctgca tggtttccag gctcctgctg
cccaaggtgc cttcctgcga ggctccggtc 600tgagcctggc ctcgggtcgg
ttcacggccc ccgtgtccgg catcttccag ttctctgcca 660gtctgcacgt
ggaccacagt gagctgcagg gcaaggcccg gctgcgggcc cgggacgtgg
720tgtgtgttct catctgtatt gagtccctgt gccagcgcca cacgtgcctg
gaggccgtct 780caggcctgga gagcaacagc agggtcttca cgctacaggt
gcaggggctg ctgcagctgc 840aggctggaca gtacgcttct gtgtttgtgg
acaatggctc cggggccgtc ctcaccatcc 900aggcgggctc cagcttctcc
gggctgctcc tgggcacgtg agggcgccca ggggggctgg 960cgaggagctg
ccgccggatc ccggggaccc tcctactgat gcccgtggtc accacaataa
1020agagccctcc accctcaaaa aaaaaaaaaa aaaaa 10559302PRTHomo sapiens
9Met Arg Arg Trp Ala Trp Ala Ala Val Val Val Leu Leu Gly Pro Gln1 5
10 15Leu Val Leu Leu Gly Gly Val Gly Ala Arg Arg Glu Ala Gln Arg
Thr 20 25 30Gln Gln Pro Gly Gln Arg Ala Asp Pro Pro Asn Ala Thr Ala
Ser Ala 35 40 45Ser Ser Arg Glu Gly Leu Pro Glu Ala Pro Lys Pro Ser
Gln Ala Ser 50 55 60Gly Pro Glu Phe Ser Asp Ala His Met Thr Trp Leu
Asn Phe Val Arg65 70 75 80Arg Pro Asp Asp Gly Ala Leu Arg Lys Arg
Cys Gly Ser Arg Asp Lys 85 90 95Lys Pro Arg Asp Leu Phe Gly Pro Pro
Gly Pro Pro Gly Ala Glu Val 100 105 110Thr Ala Glu Thr Leu Leu His
Glu Phe Gln Glu Leu Leu Lys Glu Ala 115 120 125Thr Glu Arg Arg Phe
Ser Gly Leu Leu Asp Pro Leu Leu Pro Gln Gly 130 135 140Ala Gly Leu
Arg Leu Val Gly Glu Ala Phe His Cys Arg Leu Gln Gly145 150 155
160Pro Arg Arg Val Asp Lys Arg Thr Leu Val Glu Leu His Gly Phe Gln
165 170 175Ala Pro Ala Ala Gln Gly Ala Phe Leu Arg Gly Ser Gly Leu
Ser Leu 180 185 190Ala Ser Gly Arg Phe Thr Ala Pro Val Ser Gly Ile
Phe Gln Phe Ser 195 200 205Ala Ser Leu His Val Asp His Ser Glu Leu
Gln Gly Lys Ala Arg Leu 210 215 220Arg Ala Arg Asp Val Val Cys Val
Leu Ile Cys Ile Glu Ser Leu Cys225 230 235 240Gln Arg His Thr Cys
Leu Glu Ala Val Ser Gly Leu Glu Ser Asn Ser 245 250 255Arg Val Phe
Thr Leu Gln Val Gln Gly Leu Leu Gln Leu Gln Ala Gly 260 265 270Gln
Tyr Ala Ser Val Phe Val Asp Asn Gly Ser Gly Ala Val Leu Thr 275 280
285Ile Gln Ala Gly Ser Ser Phe Ser Gly Leu Leu Leu Gly Thr 290 295
3001017PRTArtificial SequenceConsensus sequence for Treg-sTNF 10Ile
Phe Gln Phe Ser Ala Ser Leu His Val Asp His Ser Glu Leu Gln1 5 10
15Gly
* * * * *